Abstract
Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMDNterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMDNterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.
Highlights
Pyroptosis is an inflammatory type of programmed cell death that is triggered by a variety of threats, including intracellular pathogen- or host-derived perturbations of the cytosol (Bergsbaken et al, 2009; Jorgensen & Miao, 2015)
Based on the structural insights revealed using high-resolution atomic force microscopy (AFM), we derive a mechanistic model for the process of GSDMDNterm assembling transmembrane pores (Fig 6)
We here observe that independently of whether human GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5, the cleaved N-terminal domain binds and inserts into lipid membranes, where it assembles into a variety of arc, slit, and ring-shaped oligomers
Summary
Pyroptosis is an inflammatory type of programmed cell death that is triggered by a variety of threats, including intracellular pathogen- or host-derived perturbations of the cytosol (Bergsbaken et al, 2009; Jorgensen & Miao, 2015). Pyroptosis is characterized by cell swelling, cell membrane disruption, and release of cytoplasmatic contents including pro-inflammatory molecules, such as the matured form of interleukin-1b and interleukin-18. Pyroptosis is characterized by its dependence on a dedicated set of cysteinedependent proteases, the so-called inflammatory caspases, which include caspase-1, caspase-4, and caspase-5 in humans and caspase-1 and caspase-11 in mice (Cookson & Brennan, 2001; Aachoui et al, 2013). These caspases are activated by inflammasomes, multi-protein complexes that assemble upon recognizing certain cytosolic danger- or pathogen-associated molecular patterns by cytosolic receptors (Broz & Dixit, 2016). In 2015, three landmark studies identified gasderminD (GSDMD) as an essential mediator of pyroptosis in human and murine cells (He et al, 2015; Kayagaki et al, 2015; Shi et al, 2015)
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